As is known, azithromycin is a semisynthetic antibiotic belonging to the family of macrolides, whose parent is erythromycin, a natural antibiotic produced by fermentation by Streptomyces erythraeus. Macrolides are defined in general as a group of pharmacologically active molecules containing a macrolide ring, i.e. a macrocyclic lactone ring (generally having 14-16 members) to which one or more deoxy sugars can be connected. The biological activity of such compounds is linked to their ability to inhibit the biosynthesis of bacterial proteins.
The need to obtain macrolides more powerful and more chemically stable than erythromycin was the reason that led to the development of azithromycin (hereinafter also referred to by the acronym AZM). The chemical change consisted in the replacement of a keto group with a methylamino group on the lactone ring of erythromycin, so as to obtain a cyclic compound with a nitrogen atom inserted in the macrolide ring, as shown in the structural formula below:
Said chemical change improved the chemical, pharmacokinetic and microbiological profile of this macrolide with respect to the starting erythromycin. The semi-synthetic macrolide compounds which have the aforementioned substitution, named azalides, have improved pharmacokinetic properties and greater stability characteristics with respect to the starting macrolide: For this reason azithromycin is currently more widely used compared to erythromycin (Fiese, E. F., Steffen, S. H., Comparison of the acid stability of azithromycin and erythromycin A, J. Antimicrob. Chemother., January 1990, Suppl A:39-47).
Azithromycin also shows a broader spectrum of activity than the other macrolides, having a better antibacterial activity on Gram-positive bacteria, and also extending such activity to Gram-negative bacteria. In view of that azythromycin has become the drug of choice in the treatment of soft tissue infections caused by Chlamydia pneumoniae, Chlamydia trachomatis, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma hominis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Peptostreptococcus species, Streptococcus pyogenes, Streptococcus agalactiae, streptococci of groups C, F and G, Ureaplasma urealyticum, Haemophilus influenzae, etc. The pharmacokinetic profile is enhanced compared to that of erythromycin, as after oral administration a higher intracellular distribution and a longer plasma half-life ensues, thereby allowing once-daily dosing (Gladue, R. P., In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection, Antimicrob. Agents Chemother. March 1989, 33: 277-82; Ball, A. P. Azithromycin: an interim analysis, J. Int. Med. Res. November-December 1991, 19 (6):446-50). All of these factors have contributed to the success of azithromycin on erythromycin, making such active ingredient one of the antibiotic drugs most widely marketed worldwide.
Despite its excellent pharmacokinetic characteristics and the broad spectrum of action, AZM has been used, to date, mostly through the oral and parenteral routes of administration. Quite a high number of preparations based on AZM are presently on the market in the form of tablets, capsules, powders for injectable products and oral suspensions, whereas for topical ophthalmic applications azithromycin has not yet found a comparably large use. For the treatment of bacterial and trachomatous diseases affecting the ocular surface, the specialists in the field have not had available, for a long time after its introduction on the market (dating back to the early '80s), a topical ophthalmic product having AZM as active ingredient: Thus, they had to resort to oral systemic therapies to eradicate ocular infections and treat infectious diseases such as bacterial conjunctivitis, uveitis, superficial and interstitial ulcers, post-operative infections, keratitis and blepharitis (Andrews, V., Antibiotic treatment of ophthalmic infection: new developments, J. Hospital Infection 1995, 30: 268-274).
In this regard, it is known that administration through the topical ophthalmic route is preferable in order to avoid both problems of systemic absorption, and therefore toxicity, and the onset of drug resistance. This need is more urgent after the warning issued by the FDA in March 2013, on the systemic use of azithromycin in connection with cases of cardiac arrhythmias. In addition, although it is known in the literature that azithromycin following oral administration reaches significant concentrations in the lachrymal gland and conjunctiva, the available data indicate that in order to reach the MIC (Minimum Inhibitory Concentration) following topical ophthalmic administration it is nevertheless necessary to formulate the systemic AZM at high concentrations (Tabbara, K. F., Ocular level of azithromycin, Arch. Ophthalmol. 1998, 116 (12), 1625-1628; Kargioglu, Z. A., Pharmacokinetics of azithromycin in trachoma patients: serum and tear level, Ophthalmic Res. 1999, 31 (1), 47-52; I. Cochereau, I., Efficacy and safety of short duration azithromycin eye drops versus azithromycin single oral dose for the treatment of trachoma in children: a randomised, controlled, double-masked clinical trial, Br. J. Ophthalmol. 2007, 91:667-672).
The lack of AZM preparations administrable through the topical ophthalmic route is due to the physico-chemical characteristics of the azalide molecule itself. Indeed, the scientific and patent literature describes azithromycin as a molecule having a low level of solubility and a limited chemical stability in aqueous medium, which is notoriously considered to be the vehicle of choice of ophthalmic preparations. The pharmacopoeia reports that AZM is poorly soluble in water, while it is soluble in almost all organic solvents.
The first topical ophthalmic product based on AZM for the treatment of ocular bacterial infections has been placed on the market in the USA, upon approval of the FDA, in 2007, under the trade name Azasite®. This product is based on a number of patents owned by InSite Vision Inc. and licensed to Inspire Pharmaceuticals Inc. (a subsidiary of Merck & Co.), and on a patent owned by Pfizer, specifically the patents U.S. Pat. Nos. 6,159,458, 6,239,113, 6,569,443 and 7,056,893 assigned to InSite Vision and U.S. Pat. No. 6,861,411 assigned to Pfizer.
The first one of these documents is connected to some earlier patents of the same company (such as the U.S. Pat. No. 5,192,535), and concerns the production of ophthalmic release forms based on matrices of moderately crosslinked carboxylic polymers (polycarbophil), which allow to obtain a sufficiently low viscosity in order to be easily administered in the form of eye drops, and which then form a mucoadhesive gel in the eye, in order to retain the drug delivered in the site of administration for prolonged periods. The patent text refers in particular to water-soluble active ingredients, and cites pilocarpine as the preferred example.
The second and third documents (which in fact correspond to a single international patent application, Publ. No. WO00/57866 assigned to InSite Vision), concern the application of the teachings of previous patents of the same holder to the specific case of azithromycin, an active ingredient insoluble in water, which can be kept in suspension in the polymeric vehicle with delivery systems such as those mentioned above, stable from the physical point of view. The polymer delivery system proposed by the cited patents of InSite Vision is commercially known as DuraSite® (polycarbophil, disodium edetate, sodium chloride).
The Pfizer patent on which the drug Azasite® is based, U.S. Pat. No. 6,861,411, having title “Method for the treatment of eye infections with azithromycin” (and corresponding to European patent EP 0924789), reaffirms the potent antibacterial activity of azithromycin compared with erythromycin, gentamicin or other ophthalmic antibiotics, which makes it possible to implement the antibiotic therapy with a single daily administration rather than 4-6 daily doses prescribed for other ophthalmic antibiotic. The azithromycin compositions disclosed have a concentration of from 0.5 to 2.5%, and azithromycin can be formulated therein in one of the following forms:                isotonic solution at pH 7-8, in the presence of a suitable buffer (in particular borate buffer) and a tonicity adjusting agent (in particular glycerin);        in dispersed form in a matrix of white vaseline, liquid paraffin and lanolin to provide salves and ointments;        suspended in a gel thanks to the use of polyacrylic polymers, such as Carbopol.        
The third solution is actually adopted in the Azasite ophthalmic pharmaceutical product, while the other two appear to somehow represent theoretical possibilities, the first one because of the substantial insolubility of azithromycin in water, and the second due to poor patient compliance in respect of oily semisolid products such as ointments and salves for ophthalmic application. It should be noted that the text of the document does not mention any commercial preparations that can meet the stability requirements, both physical and chemical, of a pharmaceutical product, as the patent is confined to the general proposal of the therapeutic method.
The fourth US patent of InSite Vision connected to the commercial product Azasite, U.S. Pat. No. 7,056,893 of InSite Vision, finally addresses the problem of stability of the preparation of azithromycin, and proposes a pH range between 6.0 and 6.5, preferably 6.3, in which the AZM molecule is more stable. Stability studies carried out to the preservation at 25° C. and 4° C. have been reported for a period of 6 months, while for the stability studies at 40° C. are reported for up to two months, and the title of AZM is about 90% of its initial value for the best formulations, at pH 6.0 and 6.5.
From the table of stability, which can be derived from a check of the active content in the AZM formulations in DuraSite® over the 6 months, the impossibility of storing such product it at room temperature is evidente, as the formulation stored at 25° C. has a 92% of active after six months. These results highlight the need for a storage in refrigerated conditions, in order to slow down the kinetics of chemical degradation.
In a scientific work connected to the research on AZM formulations in a gelling polymer vehicle of the type of DuraSite® (Esteban, S. L., Manzo, R. H., Alovero, F. L., Azithromycin loaded on hydrogels of carbomer: chemical stability and delivery properties, Int. J. Of Pharm. 2009, 366, 53-57) the increased chemical stability of the azalide in the presence of polyacrylic polymers (Carbopol) is evidenced, compared to a formulation of AZM in phosphate buffer. In this article the greater stability of the AZM-Carbopol systems is ascribed to some electrostatic interactions between the basic functions of AZM and the acid functions present in the polymer, in agreement with what is schematically shown below:R—COOH+D→[R—COO−+DH+]wherein RCOOH represents the polyacrylic polymer, rich of carboxy moieties, and D represents the drug, having at least one basic group. This phenomenon has been interpreted by hypothesizing that a negative electrokinetic potential capable of creating a microenvironment with a pH lower than that of the solution, unfavorable to hydrolysis, is established. A similar behavior has also been observed for procaine formulated in a Carbopol gel.
The only other ophthalmic pharmaceutical product for topical use based on azithromycin is currently the eye-drops product sold in Europe under the trade name Azyter® (Laboratoires Théa), based on the European patent EP 1377316 (and other patents of the same family, including U.S. Pat. No. 7,064,109), relating to a formulation of AZM (1.5% in the preferred embodiment) in an oily vehicle formed by fatty acid triglycerides
The inventors of the patent on Azyter recall in this document the limitations associated with the use of suspensions in ophthalmology. In fact, if on one hand the precorneal retention time the of the particles of active ingredient increases, on the other hand the foreign body sensation on the ocular surface is inevitable. This unpleasant effect is more pronounced during an inflammatory process, causing by itself irritation of the ocular mucosae, in addition to uncertainty about the uniformity of dosage. According to the cited document, an ophthalmic formulation having azithromycin dissolved in an oily medium, preferably consisting of medium-chain triglycerides (MCT), solving the problem of solubility by excluding water as a vehicle, allows to avoid this drawback.
Still according to the Théa patent, the type of formulation selected also allows to increase the residence time, and therefore the precorneal bioavailability, of azalide on ocular structures, as the thin layer of “medicated oil” persists longer on the ocular surface than does an aqueous solution. In substance, in order to solve the problem related to the stability of azithromycin the inventors had to resort to the use of a non-aqueous vehicle, thanks to which Azyter is storable at 25° C. for 18 months without any need to be stored in a refrigerator.
In order to have a good compliance, an ophthalmic formulation must have a composition, in terms of aqueous vehicle, neutral pH, salt composition, value of surface tension and viscosity as similar as possible to those of the tear fluid. Therefore, it will be appreciated that an oily vehicle such as the one described in the cited patent has characteristics quite different from the optimal ones of an antibiotic in eye drops. In fact, its administration could cause blurred vision, discomfort and temporary burning sensation to the patient. If it is considered that the recipients of the therapy include pediatric patients, it will be apparent that a therapy with the eye drops proposed is not easy to manage.
As mentioned above, the main problems related to the formulation of azithromycin in water are linked to the following:
the very low water solubility of the molecule,
its susceptibility to degradation in an aqueous medium.
It has been shown that the main mechanism of degradation of azithromycin is the hydrolysis of the 1,4 α-glucosidic bond, which leads to cleavage of the sugar bound to C3 (L-cladinose) and to a microbiologically inactive metabolite (desosaminyl-azithromycin, DAZM), according to the scheme described below:

Many studies have been carried out to devise the best experimental conditions to slow down this phenomenon, and in particular both the ionic strength and the type of buffer system have been investigated, as well as the pH. Zhang and co-workers (Zhang. Y. et al., Aspect of degradation kinetics of azithromycin in aqueous solution, Chromatographia 2009, 70 (1/2, 67-73)) show that the kinetics of the hydrolytic phenomenon has the minimum value at pH 6.3 (as noted in U.S. Pat. No. 7,056,893), especially in the presence of a phosphate buffer and a low ionic strength, while it seems not to be influenced by the initial macrolide concentration. Being the degradation linked to a hydrolytic problem, and since water is the vehicle of choice of ophthalmic formulations, the difficulty linked to the problem of formulating azithromycin in an aqueous medium for a commercial preparation to be proposed for storage at 25° C. is self-evident.
In view of the foregoing, there appears to be an evident need to have available a pharmaceutical preparation in eye-drops containing azithromycin as the active ingredient, and being:                in an aqueous vehicle, stable at 25° C., and suitable for storage at room temperature for at least 24 months;        well tolerated by the patient.        